1. Field of the Invention
This invention relates to macromolecule blotting and more particularly to an improved method for electroeluting and blotting macromolecules from a chromatographic gel to an immobilizing matrix.
2. Prior Art
Smithies, Biochem. J. 61: 629-641 (1955), showed that starch gel could serve as a molecular sieve through which zone electrophoresis of proteins occurs. Since then, there have been constant innovations in the technique of gel electrophoresis. The introduction of acrylamide gels, discontinuous buffer systems, the use of sodium dodecyl sulfate (SDS) to disaggregate protein complexes to be resolved on gels, and the eventual combined use of SDS in discontinuous buffer systems for polyacrylamide gel electrophoresis have been major contributions to the development of one of the most widely used analytical and preparative tools of modern biology.
The main object of these techniques has been to visually demonstrate the homogeneity or complexity of a protein preparation by following the appearance of disappearance of a particular "band" throughout a given experimental procedure. One-dimensional gels were found to be adequate, provided only relatively simple protein samples such as viruses, bacteriophages, erythocyte ghost membranes, etc., were being analyzed. More complex systems demanded greater resolving power and new two-dimensional gel systems were developed. Today, even the thousands of polypeptides which are a part of the more intricate proteinaceous samples can be efficiently resolved.
The task of unequivocally correlating a "band" or "spot" with a recognized function has often been difficult, and this is even more so when the resolution of the proteins depends on their denaturation. Nevertheless, many approaches have been developed which allow the identification of a specific enzyme, antigen, glycoprotein or hormone receptor, etc., in a gel. These techniques rely on the ability to maintain at least one of the following prerequisites: (1) that the polypeptides retain their activity throughout electrophoresis; (2) renaturation of a denatured polypeptide; and (3) covalent crosslinkage of the protein in question to a detectable ligand prior to electrophoresis. Moreover, the actual processing of the gels entails multiple manipulations and extensive incubations and washing procedures. This is very time consuming and quite often prone to handling accidents such as breakage and tearing of wet gels or cracking during the drying of the gels.
In attempting to overcome some of the problems encountered in analyzing gels, a new approach has evolved. A number of reports have been published demonstrating that the well established approach of "Southern-blotting", i.e., transferring DNA patterns from agarose gels to nitrocellulose membrane filters, can be applied to protein patterns in polyacrylamide gels. Intact protein patterns are eluted from the gels and are immobilized on a filter substratum. The substratum is, in turn, subjected to the same type of procedures which have been used on gels for "band" or "spot" identification. However, by transferring electrophoretograms to immobilizing matrices one may benefit from the following advantages: (1) wet filters are pliable and easy to handle; (2) the immobilized proteins are readily and equally accessible to various ligands (since the limitations introduced in gels by differential porosity are obviated); (3) transfer analysis generally calls for small amounts of reagents; (4) processing times (incubations and washings) are significantly reduced; (5) multiple replicas of the gels may be made; (6) transferred patterns may be stored for months prior to their use; (7) protein transfers may undergo multiple analyses. Moreover, the transferred protein patterns are amenable to analyses which would be otherwise extremely difficult or impossible to perform on gels.
The term "blotting" today refers to the process of transferring biological macromolecules such as nucleic acids and proteins from gels to an immobilizing matrix. The term is often used in conjunction with the relevant macromolecule, e.g., protein blotting, DNA blotting and RNA blotting. The resulting filter containing transferred immobilized macromolecule is known as a "blot" or "transfer" and can be incubated with a ligand, a procedure which may be referred to as "overlay". Thus, for example, immuno-overlay, lectin overlay or calmodulin overlay refers to the incubation of a blot with an antibody, lectin or calmodulin, respectively.
In general, protein blotting should be viewed as two sequential events, namely the elution of the polypeptide from the gel and the adsorption of the eluted material to an immobilizing matrix.
Three main driving forces have been exploited for macromolecule elution. One is diffusion. Here, the gel containing the macromolecules to be transferred is sandwiched between two sheets of immobilizing matrix which are in turn sandwiched between foam pads and stainless steel screens. This final assembly is then submerged in two liters of buffer and allowed to sit for 36-48 hours. The result of this incubation is that two identical replica blots are obtained. This may or may not be an advantage. This depends on the quantity of macromolecule present and the sensitivity of the assay to be used. The efficiency of transfer may reach 75% with half the quantity available for each matrix. Since diffusion should occur in all directions loss of resolution might be expected. Because all the macromolecules in the gel are subject to the same diffusive force there is a bias in the speed of elution in favor of the lower molecular weight macromolecules.
This speed bias is a disadvantage especially when the purpose of the technique is to quantify the amount of each component in a particular sample or in comparing samples. The speed bias is also a disadvantage when subdetectable amounts of the higher molecular weight macromolecules are eluted.
The second means of macromolecule blotting is based on mass flow of buffer (convection) through the gel. This is the traditional procedure described by Southern, J. Mol. Biol. 98: 503-517 (1975). The gel is placed in a reservoir of buffer. A membrane filter is applied to the gel and paper towels are piled onto the membrane filter. The towels absorb the buffer from the reservoir through the gel and membrane filter. This movement of fluid serves as the driving force which elutes the proteins out of the gel which are then trapped in the membrane filter to create the blot. The advantages of this technique are that it takes less time (2-12 hrs.) than diffusion blotting, is more efficient, and is inexpensive since a reservoir is the only apparatus required. The major disadvantage is that this method of elution is only practical with agarose gels and is less suitable for use with polyacrylamide gels. A modification of this approach has been suggested which allows bidirectional blotting, i.e., blotting with two membrane filters, one on either side of the gel. The time for efficient solution has been dramatically reduced by applying a vacuum to facilitate the process--Peferoen, et.al., FEBS Lett. 145: 369-372 (1982).
The most widely used mode for protein blotting (it is quite often found to be advantageous in nucleic acid blotting as well) is based on electroeluting the macromolecule from the gel. The concept of electroelution of macromolecules for blotting was originally described by Arnheim and Southern, Cell 11: 363-370 (1977). Subsequently, numerous apparatus designs have been reported and some are commercially available. The essence of the technique is as follows. A wet filter material is placed on a gel making sure that no air bubbles are caught within the filter or between the filter and the gel. The filter and gel are then sandwiched between supportive porous pads such as "Scotch Brite" scouring pads, foam rubber or layers of wet blotting paper. The assembly is then supported by solid grids (usually nonconductive). It is very important that the gel and filter are firmly held together. This ensures good transfer and prevents distortion of the protein bands. The supported "gel+filter sandwich" is inserted into a tank containing "transfer buffer" and placed between two electrodes. The electrodes are connected to a power supply. Typical currents employed are in the range of 250 mA. An economical, yet efficient, design that seems to work reasonably well is that described by Bittner, et.al., Anal. Biochem. 102: 459-471 (1980).
The advantage of electroelution of macromolecules is that the time needed for elution is greatly reduced. Additionally, since the electric field strength used for elution is readily quantifiable and manipulable, the technique is conducive to the determination of exact and readily reproducible optimum transfer conditions.
Several apparatus have been reported which utilized different designs and construction for the electrodes. The design and construction of the electrode system is important because of the need as pointed out by Bittner, et.al., for a uniform electric potential, i.e., a homogeneous field across the entire surface of a chromatographic gel. A homogeneous field is needed to ensure that the macromolecules in the different lanes of the gel are uniformly transferred to the immobilizing matrix. Only then can lane to lane comparisons be made. The ideal way of designing an apparatus which would exert a homogeneous field on a chromatographic gel would be to use two parallel metal electrode plates. Because the metal electrodes would have little resistance in comparison to the buffer solution any potential applied to the electrodes would be uniformly distributed across their entire surface, thus providing a uniform electric field with which to elute the macromolecules from the slab gel. A platinum electrode is preferred because platinum is not readily degraded by electrolysis. The use of platinum foil for electrodes is impractical because of its high cost. Apparatus employing a stainless steel cathode plate with a platinum wire anode (Stellwag and Dahlberg, Nucleic Acid Research 8: 299-317 (1980), McLellan and Ramshaw, Biochemical Genetics 19: 647-654 (1981) and two graphite slabs weighing 3.75 lbs. each as anode and cathode, Gibson, Anal. Biochem. 118: 1-2 (1981) have been reported. However, operating units with these electrode designs require high current, e.g., 1.5A for two hours for the graphite slabs.
Bittner reports an apparatus employing 12 mil platinum wire for electrodes and cites indirect experimental evidence to conclude a homogeneous field is produced. The electrode is formed by stringing uninsulated platinum wire vertically 19 cm, horizontally 5.5 cm, again vertically for 19 cm, again horizontally for 5.5 cm and finally vertically for 19 cm. The distance of the two outer vertical portions of the platinum wire from the plexiglass walls of the apparatus are 2.4 cm. While it is not stated it appears from a diagram of the apparatus that the vertical portions of the wire forming the anode and cathode are aligned. The electrode assemblies are positioned very close (1.5 cm) to the gel. To infer that the electric field produced by this electrode design is uniform over the entire surface of the chromatographic gel, Bittner, et.al., compared the separation patterns of nucleic acids on 0.75% agarose gels with the separation pattern after transfer to the matrix and observed that the electroelution had occurred without distortion and with little loss of resolution. However, since nucleic acid transfers from agarose gels is readily accomplished by the technique of Southern blotting the validity of this test to conclude that a uniform electric field is generated by this electrode design is questionable. Even though the transferred patterns covered only 30% of the slab surface area they extrapolated their observation to the entire gel surface and concluded that the electrical field was uniform over the entire gel surface. Since electrode designs will produce uniform but different electric fields in different areas of a gel, the validity of extrapolating the observations from one area of a gel to the entire gel is questionable. Additionally, the fact that some loss of resolution does occur upon transfer further weakens inferences of a unifrom electric field.
The efficiency of the transfer of the individual macromolecular elements from the gel to the immobilizing matrix seems to depend on the chemical nature of the element, i.e., whether it is protein or nucleic acid, the composition of the gel and the molecular weight of the individual elements. Many researchers have reported that smaller molecular weight fragments from the electrophoretic separation of protein isolates on polyacrylamide gels are eluted with greater efficiency than larger fragments. See for example: Burnette, Anal. Biochem. 112: 195-203 (1981), Gershoni and Palade, Anal. Biochem. 124: 396-405 (1982), Howe and Hershey, J. Biol. Chem. 256 12836-12839 (1981), McLellan and Ramshaw, Biochem. Genet. 19: 647-654 (1981). This effect was documented particularly well by Howe and Hershey. By changing the immobilizing matrix every hour they were able to show that in two hours the low-molecular weight polypeptides were efficiently eluted whereas six hours were necessary to elute sufficient amounts of high molecular weight polypeptides. To illustrate contradiction in the state of the art, Bittner claims that proteins with a molecular weight range of 14,000 to 110,000 were eluted and transferred virtually quantitatively from a SDS polyacrylamide gel. Towbin, et.al., (Proc. Nat. Acad. Sci. U.S.A. 76, 4350-4354 (1979) reports quantitative transfers in urea but not in SDS polyacrylamide gels.
A number of suggestions have been made to overcome or mitigate this molecular weight bias in a transfer, among them (i) the use of reversible gel crosslinkers (Tas, et.al., Anal. Biochem. 100: 264-270 (1979) (instead of bisacrylamide), followed by gel depolymerization prior to transfer (Bolen, et.al., Appl. Environ. Microbiol. 43: 193-199 (1982), Renart, et.al., Proc. Nat. Acad. Sci. U.S.A. 76: 3116-3120 (1979)); (ii) limited protease digestion of high molecular weight proteins during electrophoretic transfer to convert them to smaller more easily elutable peptides (Gibson, Anal. Biochem. 118: 1-3 (1981)); (iii) addition of detergent SDS to "transfer buffer" to facilitate elution of high molecular weight proteins (Erickson, et.al., J. Immunol. Methods 51: 241-249 (1982). The effect of acrylamide concentration on protein elution has not been studied. One would expect that the elution of high molecular weight peptides would be affected by the porosity of the gel matrix (Gershoni and Palade, Anal. Biochem. 131: 1-15 (1983).